Chimeric genes for transforming plant cells using viral promoters

ABSTRACT

In one aspect the present invention relates to the use of viral promoters in the expression of chimeric genes in plant cells. In another aspect this invention relates to chimeric genes which are capable of being expressed in plant cells, which utilize promoter regions derived from viruses which are capable of infecting plant cells. One such virus comprises the cauliflower mosaic virux (CaMV). Two different promoter regions have been derived from the CaMV genome and ligated to heterologous coding sequences to form chimeric genes. These chimeric genes have been shown to be expressed in plant cells. This invention also relates to plant cells, plant tissue, and differentiated plants which contain and express the chimeric genes of this invention.

RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/146,621, filed Oct.28, 1993, now U.S. Pat. No. 5,352,065, which is a continuation of Ser.No. 07/625,637, filed Dec. 7, 1990, abandoned, which is a continuationof Ser. No. 06/931,492, filed Nov. 17, 1986, abandoned, which is acontinuation-in-part of Ser. No. 06/485,568 filed Apr. 15, 1983,abandoned, which is a continuation-in-part of Ser. No. 06/458,414 filedJan. 17, 1983, now abandoned.

TECHNICAL FIELD

This invention is in the fields of genetic engineering and plantbiology.

BACKGROUND ART

A virus is a microorganism comprising single or double stranded nucleicacid (DNA or RNA) contained within a protein (and possibly lipid) shellcalled a "capsid" or "coat". A virus is smaller than a cell, and it doesnot contain most of the components and substances necessary to conductmost biochemical processes. Instead, a virus infects a cell and uses thecellular processes to reproduce itself.

The following is a simplified description of how a DNA-containing virusinfects a cell; RNA viruses will be disregarded in this introduction forthe sake of clarity. First, a virus attaches to or enters a cell,normally called a "host" cell. The DNA from the virus (and possibly theentire viral particle) enters the host cell where it usually operates asa plasmid (a loop of extra-chromosomal DNA). The viral DNA istranscribed into messenger RNA, which is translated into one or morepolypeptides. Some of these polypeptides are assembled into new capsids,while others act as enzymes to catalyze various biochemical reactions.The viral DNA is also replicated and assembled with the capsidpolypeptides to form new viral particles. These viral particles may bereleased gradually by the host cell, or they may cause the host cell tolyse and release them. The released viral particles subsequently infectnew host cells. For more background information on viruses see, e.g.,Stryer, 1981 and Matthews, 1970 ( note: all references cited herein,other than patents, are listed with citations after the examples).

As used herein, the term "virus" includes phages and viroids, as well asreplicative intermediates. As used herein, the phrases "viral nucleicacid" and DNA or RNA derived from a virus" are construed broadly toinclude any DNA or RNA that is obtained or derived from the nucleic acidof a virus. For example, a DNA strand created by using a viral RNAstrand as a template, or by chemical synthesis to create a knownsequence of bases determined by analyzing viral DNA, would be regardedas viral nucleic acid.

The host range of any virus (i.e., the variety of cells that a type ofvirus is capable of infecting) is limited. Some viruses are capable ofefficient infection of only certain types of bacteria; other viruses caninfect only plants, and may be limited to certain genera; some virusescan infect only mammalian cells. Viral infection of a cell requires morethan mere entry of the viral DNA or RNA into the host cell; viralparticles must be reproduced within the cell. Through various assays,those skilled in the art can readily determine whether any particulartype of virus is capable of infecting any particular genus, species, orstrain of cells. As used herein, the term "plant virus" is used todesignate a virus which is capable of infecting one or more types ofplant cells, regardless of whether it can infect other types of cells.

With the possible exception of viroids (which are poorly understood atpresent), every viral particle must contain at least one gene which canbe "expressed" in infected host cells. The expression of a gene requiresthat a segment of DNA or RNA must be transcribed into or function as astrand of messenger RNA (mRNA), and the mRNA must be translated into apolypeptide. Most viruses have about 5 to 10 different genes, all ofwhich are expressed in a suitable host cell.

In order to be expressed in a cell, a gene must have a promoter which isrecognized by certain enzymes in the cell. Gene promoters are discussedin some detail in the parent application Ser. No. 458,414 cited above,the contents of which are incorporated herein by reference. Thoseskilled in the art recognize that the expression of a particular gene toyield a polypeptide is dependent upon two distinct cellular processes. Aregion of the 5' end of the gene called the promoter, initiatestranscription of the gene to produce a mRNA transcript. The mRNA is thentranslated at the ribosomes of the cell to yield an encoded polypeptide.Therefore, it is evident that although the promoter may functionproperly, ultimate expression of the polypeptide depends at least inpart on post-transcriptional processing of the mRNA transcript.

Promoters from viral genes have been utilized in a variety of geneticengineering applications. For example, chimeric genes have beenconstructed using various structural sequences (also called codingsequences) taken from bacterial genes, coupled to promoters taken fromviruses which can infect mammalian cell (the most commonly usedmammalian viruses are designated as Simian Virus 40 (SV40) and HerpesSimplex Virus (HSV)). These chimeric genes have been used to transformmammalian cells. See, e.g., Mulligan et al 1979; Southern and Berg 1982.In addition, chimeric genes using promoters taken from viruses which caninfect bacterial cells have been used to transform bacterial cells; see,e.g., the phage lambda P_(L) promoter discussed in Maniatis et al, 1982.

Several researchers have theorized that it might be possible to utilizeplant viruses as vectors for transforming plant cells. See, e.g., Hohnet al, 1982. In general, a "vector" is a DNA molecule useful fortransferring one or more genes into a cell. Usually, a desired gene isinserted into a vector, and the vector is then used to infect the hostcell.

Several researchers have theorized that it might be possible to createchimeric genes which are capable of being expressed in plant cells, byusing promoters derived from plant virus genes. See, e.g., Hohn et al,1982, at page 216.

However, despite the efforts of numerous research teams, prior to thisinvention no one had succeeded in (1) creating a chimeric genecomprising a plant virus promoter coupled to a heterologous structuralsequence and (2) demonstrating the expression of such a gene in any typeof plant cell.

CAULIFLOWER MOSAIC VIRUS (CaMV)

The entire DNA sequence of CaMV has been published. Gardner et al, 1981;Hohn et al, 1982. In its most common form, the CaMV genome is about 8000bp long. However, various naturally occurring infective mutants whichhave deleted about 500 bp have been discovered; see Howarth et al 1981.The entire CaMV genome is transcribed into a single mRNA, termed the"full-length transcript" having a sedimentation coefficient of about35S. The promoter for the full-length mRNA (hereinafter referred to as"CaMV(35S)") is located in the large intergenic region about 1 kbcounterclockwise from Gap 1 (see Guilley et al, 1982).

CaMV is believed to generate at least eight proteins; the correspondinggenes are designated as Genes I through VIII. Gene VI is transcribedinto mRNA with a sedimentation coefficient of 19S. The 19S mRNA istranslated into a protein designated as P66, which is an inclusion bodyprotein. The 19S mRNA is promoted by the 19S promoter, located about 2.5kb counterclockwise from Gap 1.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to the use of viralpromoters in the expression of chimeric genes in plant cells. In anotheraspect this invention relates to chimeric genes which are capable ofbeing expressed in plant cells, which utilize promoter regions derivedfrom viruses which are capable of infecting plant cells. One such viruscomprises the cauliflower mosaic virus (CaMV). Two different promoterregions have been derived from the CaMV genome and ligated toheterologous coding sequences to form chimeric genes. These chimericgenes have been proven to be expressed in plant cells. This inventionalso relates to plant cells, plant tissue (including seeds andpropagules), and differentiated plants which have been transformed tocontain viral promoters and express the chimeric genes of thisinvention, and to polypeptides that are generated in plant cells by thechimeric genes of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures herein are schematic representations; they have not beendrawn to scale.

FIG. 1 represents the creation and structure of plasmid pMON93.

FIG. 2 represents the creation and structure of plasmid pMON156.

FIG. 3 represents the creation and structure of plasmid pMON110.

FIG. 4 represents the creation and structure of plasmid pMON132.

FIG. 5 represents the creation and structure of plasmid pMON155.

FIG. 6 represents the creation and structure of plasmid pMON81.

FIG. 7 represents the creation and structure of plasmid pMON125.

FIG. 8 represents the creation and structure of plasmid pMON172.

FIG. 9 represents the creation and structure of phage M12.

FIG. 10 represents the creation and structure of plasmids pMON183 andpMON184.

DETAILED DESCRIPTION OF THE INVENTION

In one preferred embodiment of this invention, a chimeric gene wascreated which contained the following elements:

1. a promoter region and a 5' non-translated region derived from theCaMV (19S) gene, which codes for the P66 protein;

2. a partial coding sequence from the CaMV (19S) gene, including an ATGstart codon and several internal ATG sequences, all of which were in thesame frame as a TGA termination sequence immediately inside the desiredATG start codon of the NPTII gene;

3. a structural sequence derived from a neomycin phosphotransferase II(NPTII) gene; this sequence was preceded by a spurious ATG sequence,which was in the same reading frame as a TGA sequence within the NPTIIstructural sequence; and,

4. a 3' non-translated region, including a poly-adenylation signal,derived from a nopaline synthase (NOS) gene.

This chimeric gene, referred to herein as the CaMV(19S)-NPTII-NOS gene,was inserted into plasmid pMON120 (described in the parent application,Ser. No. 458,414; ATCC accession number 39263) to create a plasmiddesignated as pMON156. Plasmid pMON156 was inserted into anAgrobacterium tumefaciens cell, where it formed a co-integrate Tiplasmid by means of a single crossover event with a Ti plasmid in the A.tumefaciens cell, using a method described in the parent application.The chimeric gene in the co-integrate plasmid was within a modifiedT-DNA region in the Ti plasmid, surrounded by left and right T-DNAborders.

A. tumefaciens cells containing the co-integrate Ti plasmids with theCaMV(19S)-NPTII-NOS genes were used to infect plant cells, using amethod described in the parent application. Some of the plant cells weregenetically transformed, causing them to become resistant to anantibiotic (kanamycin) at concentrations which are toxic tountransformed plant cells.

A similar chimeric gene was created and assembled in a plasmiddesignated as pMON155. This chimeric gene resembled the gene in pMON156,with two exceptions:

1. an oligonucleotide linker having stop codons in all three readingframes was inserted between the CaMV(19S) partial structural sequenceand the NPTII structural sequence; and,

2. the spurious ATG sequence on the 5' side of the NPTII structuralsequence was deleted.

The construction of this chimeric gene is described in Example 2. Thisgene was inserted into A. tumefaciens cells and subsequently into plantcells. Its level of expression was apparently higher than the expressionof the similar gene in pMON156, as assayed by growth on higherconcentrations of kanamycin.

CREATION OF PLASMIDS pMON183 and 184; CaMV(35S)

In an alternate preferred embodiment of this envention, a chimeric genewas created comprising

(1) a promoter region which causes transcription of the 35S mRNA ofcauliflower mosaic virus, CaMV(35S);

(2) a structural sequence which codes for NPTII; and

(3) a nopaline synthase (NOS) 3' non-translated region.

The assembly of this chimeric gene is described in Example 3. This genewas inserted into plant cells and it caused them to become resistant tokanamycin.

Petunia plants cannot normally be infected by CaMV. Those skilled in theart may determine through routine experimentation whether any particularplant viral promoter (such as the CaMV promoter) will function atsatisfactory levels in any particular type of plant cell, includingplant cells that are outside of the normal host range of the virus fromwhich the promoter was derived.

It is possible to regenerate genetically transformed plant cells intodifferentiated plants. One method for such regeneration was described inU.S. patent application entited "Genetically Transformed Plants", Ser.No. 458,402. That application was filed simultaneously with, andincorporated by reference into, the parent application of thisinvention. The methods of application Ser. No. 458,402 may be used tocreate differentiated plants (and their progeny) which contain andexpress chimeric genes having plant virus promoters.

It is possible to extract polypeptides generated in plant cells bychimeric genes of this invention from the plant cells, and to purifysuch extracted polypeptides to a useful degree of purity, using methodsand substances known to those skilled in the art.

Those skilled in the art will recognize, or may ascertain using no morethan routine experimentation, numerous equivalents to the specificembodiments described herein. Such equivalents are within the scope ofthis invention, and are covered by the claims below.

EXAMPLES EXAMPLE 1 Creation and Use of pMON156

Plasmids which contained CaMV DNA were a gift to Monsanto Company fromDr. R. J. Shepherd, University of California, Davis. To the best ofApplicants' knowledge and belief, these plasmids (designated as pOS1)were obtained by inserting the entire genome of a CaMV strain designatedas CM4-184 (Howarth et al, 1981) into the Sal I restriction site of apBR322 plasmid (Bolivar et al, 1978). E. coli cells transformed withpOS1 were resistant to ampicillin (Amp^(R)) and sensitive totetracycline (Tet^(S)).

Various strains of CaMV suitable for isolation of CaMV DNA which can beused in this invention are publicly available; see, e.g., ATCC Catalogueof Strains II, p. 387 (3rd edition, 1981).

pOS1 DNA was cleaved with HindIII. Three small fragments were purifiedafter electrophoresis on an 0.8% agarose gel using NA-45 membrane(Schleicher and Schuell, Keene NH). The smallest fragment, about 500 bpin size, contains the 19S promoter. This fragment was further purifiedon a 6% acrylamide gel. After various manipulations which did not changethe sequence of this fragment (shown in FIG. 1), it was digested withMboI to created 455 bp HindIII-MboI fragment. This fragment was mixedwith a 1250 bp fragment obtained by digesting pMON75 (described andshown in FIG. 9 of the parent application Ser. No. 458,414) with BglIIand EcoRI. This fragment contains the NPTII structural sequence and theNOS 3' non-translated region. The two fragments were ligated by theircompatible MboI and BglII overhangs to create a fragment containing theCaMV(19S)-NPTII -NOS chimeric gene. This fragment was inserted intopMON120 (described and shown in FIG. 10 of the parent application, Ser.No. 458,414; ATCC accession number 39263) which had been cleaved withHindIII and EcoRI. The resulting plasmid was designated as pMON156, asshown in FIG. 2.

Plasmid pMON156 was inserted into E. coli cells and subsequently into A.tumefaciens cells where it formed a co-integrate Ti plasmid having theCaMV(19S)-NPTII-NOS chimeric gene surrounded by T-DNA borders. A.tumefaciens cells containing the co-integrate plasmids wereco-cultivated with petunia cells. The foregoing methods are described indetail in a separate application, entitled "Plasmids for TransformingPlant Cells" Ser. No. 458,411, which was filed simultaneously with andincorporated by reference into parent application, Ser. No. 458,414.

The co-cultivated petunia cells were cultured on media containingkanamycin, an antibiotic which is toxic to petunia cells. Kanamycin isinactivated by the enzyme NPTII, which does not normally exist in plantcells. Some of the co-cultivated petunia cells survived and producedcolonies on media containing up to 50 ug/ml kanamycin. This indicatedthat the CaMV(19S )-NPTII-NOS genes were expressed in petunia cells.These results were confirmed by Southern blot analysis of transformedplant cell DNA.

EXAMPLE 2 Creation of pMON155

Plasmid pMON72 was obtained by inserting a 1.8 kb HindIII-BamHI fragmentfrom bacterial transposon Tn5 (which contains an NPTII structuralsequence) into a PstI⁻ pBR327 plasmid digested with HindIII and BamHI.This plasmid was digested with BglII and PstI to remove the NPTIIstructural sequence.

Plasmid pMON1001 (described and shown in FIG. 6 of the parentapplication) from dam⁻ cells was digested with BglII and PstI to obtaina 218 bp fragment with a partial NPTII structural sequence. Thisfragment was digested with MboI to obtain a 194 bp fragment.

A triple ligation was performed using (a) the large PstI-BglII fragmentof pMON72; (b) PstI-MboI fragment from pMON1001; and (c) a syntheticlinker with BglII and MboI ends having stop codons in all three readingframes. After transformation of E. coli cells and selection forampicillin resistant colonies, plasmid DNA from Amp^(R) colonies wasanalyzed. A colony containing a plasmid with the desired structure wasidentified. This plasmid was designated pMON110, as shown on FIG. 3.

In order to add the 3' end of the NPTII structural sequence to the 5'portion in pMON110, pMON110 was treated with XhoI. The resultingoverhanging end was filled in to create a blunt end by treatment withKlenow polymerase and the four deoxy-nucleotide triphosphates (dNTP's),A, T, C, and G. The Klenow polymerase was inactivated by heat, thefragment was digested with PstI, and a 3.6 kb fragment was purified.Plasmid pMON76 (described and shown in FIG. 9 of the parent application)was digested with HindIII, filled in to create a blunt end with Klenowpolymerase and the four dNTP's, and digested with PstI. An 1100 bpfragment was purified, which contained part of the NPTII structuralsequence, and a nopaline synthase (NOS) 3' non-translated region. Thisfragment was ligated with the 3.6 kb fragment from pMON110. The mixturewas used to transform E. coli cells; Amp R cells were selected, and acolony having a plasmid with the desired structure was identified. Thisplasmid was designated pMON132, as shown on FIG. 4. Plasmid pMON93(shown on FIG. 1) was digested with HindIII, and a 476 bp fragment wasisolated. This fragment was digested with MboI, and a 455 bpHindIII-MboI fragment was purified which contained the CaMV (19S)promoter region, and 5' non-translated region. Plasmid pMON132 wasdigested with EcoRI and BglII to obtain a 1250 bp fragment with (1) thesynthetic linker equipped with stop codons in all three reading frames;(2) the NPTII structural sequence; and (3) the NOS 3 ' non-translatedregion. These two fragments were joined together through the compatibleMboI nd BglII ends to create a CaMV (19S)-NPTII-NOS chimeric gene.

This gene was inserted into pMON120, which was digested with HindIII andEcoRI, to create plasmid pMON155, as shown in FIG. 5.

Plasmid pMON155 was inserted into A. tumefaciens GV3111 cells containinga Ti plasmid, pTiB6S3. The pMON155 plasmid formed a cointegrate plasmidwith the Ti plasmid by means of a single crossover event. Cells whichcontain this co-integrate plasmid have been deposited with the AmericanType Culture Center, and have been assigned ATCC accession number 39336.A fragment which contains the chimeric gene of this invention can beobtained by digesting the co-integrate plasmid with HindIII and EcoRI,and purifying the 1.7 kb fragment. These cells have been used totransform petunia cells, allowing the petunia cells to grow on mediacontaining at least 100 ug/ml kanamycin.

EXAMPLE 3 Creation of pMON183 and 184

Plasmid pOS1 (described in Example 1) was digested with BglII, and a1200 bp fragment was purified. This fragment contained the 35S promoterregion and part of the 5' non-translated region. It was inserted intoplasmid pSHL72 which had been digested with BamHI and BglII (pSHL72 isfunctionally equivalent to pAGO60, described in Colbere-Garapin et al,1981). The resulting plasmid was designated as pMON50, as shown on FIG.6.

The cloned BglII fragment contains a region of DNA that acts as apolyadenylation site for the 35S RNA transcript. This polyadenylationregion was removed as follows: pMON50 was digested with AvaII and an1100 bp fragment was purified. This fragment was digested with EcoRI*and EcoRV. The resulting 190 bp EcoRV-EcoRI* fragment was purified andinserted into plasmid pBR327, which had been digested with EcoRI* andEcoRV. The resulting plasmid, pMON81, contains the CaMV 35S promoter ona 190 bp EcoRV-EcoRI* fragment, as shown in FIG. 6.

To make certain the entire promoter region of CaMV(35S) was present inpMON81, a region adjacent to the 5' (EcoRV) end of the fragment wasinserted into pMON81 in the following way. Plasmid pMON50 prepared fromdam⁻ cells was digested with EcoRI and BglII and the resultant 1550 bpfragment was purified and digested with MboI. The resulting 725 bp MboIfragment was purified and inserted into the unique BglII site of plasmidpKC7 (Rao and Rogers, 1979) to give plasmid pMON125, as shown in FIG. 7.The sequence of bases adjacent to the two MboI ends regenerates BglIIsites and allows the 725 bp fragment to be excised with BglII.

To generate a fragment carrying the 35S promoter, the 725 bp BglIIfragment was purified from pMON125 and was subsequently digested withEcoRV and AluI to yield a 190 bp fragment. Plasmid pMON81 was digestedwith BamHI, treated with Klenow polymerase and digested with EcoRV. The3.1 kb EcoRV-BamHI (blunt) fragment was purified, mixed with the 190 bpEcoRV-AluI fragment and treated with DNA ligase. Followingtransformation and selection of ampicillin-resistant cells, plasmidpMON172 was obtained which carries the CaMV(35S) promoter sequence on a380 bp BamHI-EcoRI fragment, as shown on FIG. 8. This fragment does notcarry the polyadenylation region for the 35S RNA. Ligation of the AluIend to the filled-in BamHI site regenerates the BamHI site.

To rearrange the restriction endonuclease sites adjacent to theCaMV(35S) promoter, the 380bp BamHI-EcoRI fragment was purified frompMON172, treated with Klenow polymerase, and inserted into the uniqueSinai site of phage M13 top8. One recombinant phage, M12, carried the380 bp fragment in the orientation shown on FIG. 9. The replicative formDNA from this phage carries the 35S promoter fragment on an EcoRI(5')-BamHI (3') fragment, illustrated below. ##STR1##

Plasmids carrying a chimeric gene CaMV(35S) promoter region-NPTIIstructural sequence-NOS 3' non-translated region) were assembled asfollows. The 380 bp EcoRI-BamHI CaMV(35S) promoter fragment was purifiedfrom phage M12 RF DNA and mixed with the 1250 bp BglII-EcoRI NPTII-NOSfragment from pMON75. Joining of these two fragments through theircompatible BamHI and BglII ends results in a 1.6 kb CaMV(35S)-NPTII-NOSchimeric gene. This gene was inserted into pMON120 at the EcoRI site inboth orientations. The resultant plasmids, pMON183 and 184, appear inFIG. 10. These plasmids differ only in the direction of the chimericgene orientation.

These plasmids were used to transform petunia cells, as described inExample 1. The transformed cells are capable of growth on mediacontaining 100 ug/ml kanamycin.

COMPARISON OF CaMV(35S) AND NOS PROMOTERS

Chimeric genes carrying the nopaline synthase (NOS) promoter or thecauliflower mosaic virus full-length transcript promoter (CaMV(35S))were constructed. In both cases, the promoters, which contain theirrespective 5' non-translated regions were joined to a NPTII codingsequence in which the bacterial 5' leader had been modified so that aspurious ATG translational initiation signal (Southern and Berg, 1982)has been removed.

Plasmid pMON200 is a derivative of previously described intermediatevector pMON120 (ATCC accession number 39263). pMON200 contains amodified chimeric nopaline synthase-neomycin phosphotransferasenopalinesynthase gene (NOS/NPTII/NOS) which confers kanamycin (Km^(R))resistance to the transformed plant. The modified chimeric Km^(R) genelacks an upstream ATG codon present in the bacterial leader sequence anda synthetic multilinker with unique HindIII, XhoI, BglII, XbaI, ClaI andEcoRI restriction sites.

Plasmid pMON273 is a derivative of pMON200 in which the nopalinesynthase promoter of the chimeric NOS-NPTII-NOS gene has been replacedwith the CaMV(35S) promoter.

The CaMV(35S) promoter fragment was isolated from plasmid pOS-1, aderivative of pBR322 carrying the entire genome of CM4-184 as a SalIinsert (Howarth et al., 1981). The CM4-184 strain is a naturallyoccurring deletion mutant of strain CM1841. The nucleotide sequence ofthe CM1841 (Gardner et al., 1981) and Cabb-S (Franck et al., 1980)strains of CaMV have been published as well as some partial sequence fora different CM4-184 clone (Dudley et al., 1982). The nucleotidesequences of the 35S promoter regions of these three isolates areessentially identical. In the following the nucleotide numbers reflectsthe sequence of Gardner et al. (1981). The 35S promoter was isolated asan AluI (n 7143)-EcoRI* (n 7517) fragment which was inserted first intopBR322 cleaved with BamHI, treated with the Klenow fragment of DNApolymerase I and then cleaved with EcoRI. The promoter fragment was thenexcised from pBR322 with BamHI and EcoRI, treated with Klenow polymeraseand inserted into the Sinai site of M13 mp8 so that the EcoRI site ofthe mp8 multilinker was at the 5' end of the promoter fragment. Sitedirected mutagenesis (Zoller and Smith, 1982) was then used to introducea G at nucleotide 7464 to create a BglII site. The 35S promoter fragmentwas then excised from the M13 as a 330 bp EcoRI-BglII site. The 35Spromoter fragment was then excised from the M13 as a 330 bp EcoRI-BglIIfragment which contains the 35S promoter, 30 nucleotides of the 5'non-translated leader but does not contain any of the CaMV translationalinitiators nor the 35S transcript polyadenylation signal that is located180 nucleotides downstream from the start of transcription (Covey etal., 1981; Guilley et al., 1982). The CaMV(35S) promoter sequencedescribed above is listed below. ##STR2##

The 35S promoter fragment was joined to a 1.3 kb BglII-EcoRI fragmentcontaining the Tn5 neomycin phosphotrans ferase II coding sequencemodified so that the translational initiator signal in the bacterialleader sequence had been removed and the NOS 3' non-translated regionand inserted into pMON120 to give pMON273.

These plasmids were transferred in E. coli strain JM101 and then matedinto Agrobacterium tumefaciens strain GV3111 carrying the disarmedpTiB6S3-SE plasmid as described by Fraley et al. (1983).

Plant Transformation

Cocultivation of Petunia protoplasts with A. tumefaciens, selection ofkanamycin resistant transformed callus and regeneration of transgenicplants was carried out as described in Fraley et al. (1984).

Preparation of DNAs

Plant DNA was extracted by grinding the frozen tissue in extractionbuffer (50mM TRIS-HCl pH 8.0, 50 mM EDTA, 50 mM NaCl, 400 ul/ml EtBr, 2%sarcosyl). Following low speed centrifugation, cesium chloride was addedto the supernatant (0.85 gm/ml). The CsCl gradients were centrifuged at150,000×g for 48 hours. The ethidium bromide was extracted withisopropanol, the DNA was dialyzed, and ethanol precipitated.

Southern Hybridization Analysis

10 ug of each plant DNA was digested, with BamHI for pMON200 plant DNAsand EcoRI for pMON273 plant DNAs. The fragments were separated byelectrophoresis on a 0.8% agarose gel and transferred to nitrocellulose(Southern, 1975). The blots were hybridized (50% formamide, 3×SSC, 5×denhardt's, 0.1% SDS and 20 ug/ml tRNA) with nick-translated pMON273plasmid DNA for 48-60 hours at 42° C.

Preparation of RNA from Plant Tissue

Plant leaves were frozen in liquid nitrogen and ground to a fine powderwith a mortar and pestle. The frozen tissue was added to a 1:1 mixtureof grinding buffer and PCE (1% Tri-iso-propylnaphtalenesulfonic acid, 6%p-Aminosalicylic acid, 100 mM NaCl, 1% SDS and 50 mM 2-mercaptoethanol;PCI [phenol: chloroform: isoamyl alcohol (24:24:1)] and homogenizedimmediately with a polytron. The crude homogenate was mixed for 10 minand the phases separated by centrifugation. The aqueous phase then wasre-extracted with an equal volume of PCI. The aqueous phase was ethanolprecipitated with one tenth volume of 3M NaAcetate and 2.5 volumes ofethanol. The nucleic acid pellet was resuspended in water. An equalvolume of 4M lithium chloride LiCl was added and the mix was placed onice for 1 hour or overnight. Following centrifugation, the pellet wasresuspended in water the LiCl precipitation repeated 3 times. The finalLiCl pellet was resuspended in water and ethanol precipitated.

Poly (A) containing RNA was isolated by passing total RNA over an Oligod(T) cellulose Type III (Collaborative Research) column. Quantitation ofthe poly (A) containing RNA involved annealing an aliquot of the RNA toradio-labeled poly U [(uridylate 5,6-3H)-polyuridylic acid] (New EnglandNuclear), followed by RNase A treatment (10 ug per ml for 30 minutes at37° C.). The reaction mix was spotted on DE-81 filter paper, washed 4×with 0.5M NaPhosphate (pH 7.5) and counted. Globin poly (A) containingRNA (BRL) was used as a standard.

Northern Hybridization Analysis

5 ug of poly (A) RNA from each plant source was treated with glyoxal anddimethysulfoxide (Maniatis, 1982). The RNAs were electrophoresed in 1.5%agarose gels (0.01M NaH₂ PIPO₄, pH 6.5) for 7 hours at 60 volts. Theglyoxylated RNAs were electroblotted (25 mM NaH₂ PO₄ /NaHPO₄, pH 6.5)for 16 hours at 125 amps from the gel to GeneScreen® (New EnglandNuclear). The filters were hybridized as per manufacturer's instructions(50% formamide, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin,0.02% ficoll, 5×SSC, 1.0% SDS, 100 u/ml tRNA and probe) for 48-60 hoursat 42° C. with constant shaking. The nick-translated DNAs used as probeswere the 1.3 kb BglII/EcoRI NPTII fragment purified from the pMON273plasmid for detecting the NPTII transcript, and the petunia smallsubunit gene as an internal standard for comparing the amount of RNA perlane. The membranes were washed 2×100 ml of 2×SSC at room temperaturefor 5 minutes 2×100 ml of 2×SSC/10% SDS at 65° C. for 30 minutes. Themembranes were exposed to XAR-5 film with a DuPont intensifying screenat -80° C.

Neomycin Phosphotransferase Assay

The gel overlay assay was used to determine the steady state level ofNPTII enzyme activity in each plant. Several parameters wereinvestigated for optimizing the sensitivity of the assay in planttissue. Early observations showed that the level of NPTII activityvaried between leaves from different positions on the same plant. Thisvariability was minimized when the plant extract was made from pooledtissue. A paper hole punch was used to collect 15 disks from both youngand old leaves. Grinding the plant tissue in the presence of micro-beads(Ferro Corp) rather than glass beads increased the plant protein yield4-fold.

To optimize detection of low levels of NPTII activity a saturation curvewas prepared with 10-85 ug/lane of plant protein. For the pMON200 (NOS)plants, NPTII activity was not detectable at less than 50 ug/lane oftotal protein (2 hour exposure) while activity was detectable at 20ug/lane for the pMON273 plants. There was a non-linear increase in NPTIIactivity for pMON200 NOS plants between 40 and 50 ug of protein perlane. This suggested that the total amount of protein may affect thestability of the NPTII enzyme. Supplementing plant cell extracts with30-45 ug per lane of bovine serum albumin (BSA), resulted in a linearresponse; NPTII activity increased proportionately as plant proteinlevels increased. The addition of BSA appears to stabilize the enzyme,resulting in a 20-fold increase in the sensitivity of the assay.Experiments indicate that 25 ug/lane of pMON273 plant protein and 70ug/lane of pMON200 plant protein was within the linear range of theassay in the presence of BSA. Elimination of SDS from the extractionbuffer resulted in a 2-fold increase in assay sensitivity. Leaf diskswere pooled from each plant for the assay. The tissue was homogenizedwith a glass rod in a microfuge tube with 150-200 ul of extractionbuffer (20% glycerol, 10% β-mercaptoethanol, 125 mM Tris-HCl pH 6.8, 100ug/ml bromophenol blue and 0.2% SDS). Following centrifugation in amicrofuge for 20 minutes, total protein was determined using theBradford assay. 25 ug of pMON273/3111SE plant protein or 70 ug ofpMON200/3111SE plant protein, supplemented with BSA, was loaded on anative polyacrylamide gel as previously described. The polyacrylamidegel was equilibrated for 30 minutes in water and then 30 minutes inreaction buffer (67 mM TRI S-maleate pH 7.1, 43 mM MgCl₂, 400 mM NH₄Cl), transferred onto a glass plate, and overlaid with a 1.5% agarosegel. The overlay gel contained the neomycin phosphotransferasesubstrates: 450 uCi [γ-³² ] ATP and 27 ug/ml neomycin sulfate (Sigma).After 1 hour at room temperature a sheet of Whatman P81 paper, twosheets of Whatman 3MM paper, a stack of paper towels and a weight wereput on top of the agarose gel. The phosphorylated neomycin is positivelycharged and binds to the P81 phosphocellulose ion exchange paper. Afterblotting overnight, the P81 paper was washed 3× in 80° C. water,followed by 7 room temperature washes. The paper was air dried andexposed to XAR-5 film. Activity was quantitated by counting the ³²P-radioactivity in the NPTII spot. The NPTII transcript levels andenzyme activities in two sets of transgenic petunia plants werecompared. In one set of plants (pMON273) the NPTII coding sequence ispreceded by the CaMV(35S) promoter and leader sequences, in the otherset of plants (PMON200) the NPTII coding region is preceded by thenopaline synthase promoter and leader sequences. The data indicates thepMON273 plants contain about a 30 fold greater level of NPTII transcriptthan the pMON200 plants, see Table I below.

                  TABLE I                                                         ______________________________________                                        QUANTITATION OF NPTII TRANSCRIPT LEVELS                                       AND NPTII ACTIVITY IN pMON273 AND pMON200                                     PLANTS                                                                                      Relative  Relative                                              Plant         NPTII     NPTII                                                 Number        Transcript.sup.a                                                                        Activity.sup.b                                        ______________________________________                                        pMON 273                                                                      3272          682       113                                                   3271          519       1148                                                  3349          547       447                                                   3350          383       650                                                   3343          627       1539                                                  Average       551       779                                                   pMON 200                                                                      2782           0        0.22                                                  2505           0        5.8                                                   2822           0        0                                                     2813           34       19                                                    2818           0        1.0                                                   3612           45       0.33                                                  2823           97       23                                                    Average        19       7                                                                   ˜30-fold                                                                          ˜110-fold                                                     difference                                                                              difference                                            ______________________________________                                         .sup.a Numbers derived from silver grain quantitation of autoradiogram.       The RNA per lane was determined by filter hybridization to a petunia smal     subunit gene. The NPTII transcript values obtained with the NPTII probe       were normalized for the amount of RNA in each lane.                           .sup.b Numbers represent quantitation of NPT assay. Values were obtained      by scintillation counting of 32P-NPTII spots on the PE81 paper used in th     NPT assay as previously described. Values have been adjusted for the          different amounts of protein loaded on the gels (25 ug) for pMON273 and 7     ug for MON200 plants).                                                   

Consistent with this observation is the finding that the pMON273 leafextracts have higher NPTII enzyme activity than the pMON200 leafextracts. In several of the transgenic plants, there is a substantialvariation in both RNA and enzyme levels which cannot be accounted for bythe slight difference in gene copy number. Such "position effects" havebeen reported in transgenic mice and fruit flies and have not yet beenadequately explained at the molecular level. Although, there is not aclear correlation between insert copy number and level of chimeric geneexpression, the fact that 4 of the 7 pMON200 transgenic plants contain 2copies of the NOS-NPTII-NOS gene would suggest that the differentialexpression of the CaMV(35S) promoter is actually slightly underestimatedin these studies.

The constructs described in this comparative example have identicalcoding regions and 3' non-translated regions, indicating that thedifferences in the steady state transcript levels of these chimericgenes is a result of the 5' sequences.

COMPARISON OF CAMV19S AND CaMV(35S) PROMOTERS

Chimeric genes were prepared comprising either the CaMV19S or CaMV(35S)promoters. As in the above example, the promoters contained theirrespective 5' non-translated regions and were joined to a NPTII codingsequence in which the bacterial 5' leader had been modified to remove aspurious ATG translational initiation signal. The constructs tested werepMON203 and pMON204 containing the CaMV19S/NPTII/NOS gene and pMON273containing the CaMV(35S)/NPTII/NOS gene.

Construction of pMON203

The CaMV 19S promoter fragment was isolated from plasmid pOS-1, aderivative of pBR322 carrying the entire genome of CM4-184 as a SalIinsert (Howarth et al., 1981). The CM4-184 strain is a naturallyoccurring deletion mutant of strain CM1841. The references to nucleotidenumbers in the following discussion are those for the sequence of CM1841(Gardner et al., 1981). A 476 bp fragment extending from the HindIIIsite at bp 5372 to the HindIII site at bp 5848 was cloned into M13 mp8for site directed mutagenesis (Zoller and Smith, 1982) to insert an XbaI(5'-TCTAGA) site immediately 5' of the first ATG translationalinitiation signal in the 19S transcript (Dudley et al., 1982 ). Theresulting 400 bp HindIII-XbaI fragment was isolated and joined to the1.3 kb XbaI-EcoRI fragment of pMON273 which carries the neomycinphosphotransferase II (NPTII') coding sequence modified so that theextra ATG translational initiation signal in the bacterial leader hadbeen removed and the nopaline synthase 3' nontranslated region (NOS).The resulting 1.7 kb HindIII-EcoRI fragment was inserted into pMON120between the EcoRI and HindIII sites to give pMON203. The completesequence of the 19S promoter-NPTII leader is given below. ##STR3##

Construction of pMON204

The 400 bp HindIII-XbaI fragment containing the CaMV19S promoter wasjoined to a synthetic linker with the sequence: ##STR4## to add a BglIIsite to the 3' end of the promoter fragment. The HindIII-BglII fragmentwas joined to the 1.3 kb BglII-EcoRI fragment of pMON128 that containsthe natural, unmodified NPTII coding sequence joined to the NOS 3'nontranslated signals and inserted into the EcoRI and HindIII sites ofpMON120. The resulting plasmid is pMON204. The CaMV 19S promoter signalsin this plasmid are identical to those in pMON203. The only differenceis the sequence of the 5' nontranslated leader sequence which in pMON204contains the extra ATG signal found in the bacterial leader of NPTII andcontains extra bases from the synthetic linker and bacterial leadersequence.

Petunia leaf discs were transformed and plants regenerated as describedabove. The gel overlay assay was used to determine NPTII levels intransformants.

Quantitation was done by scintillation counting of ³² P-neomycin, theend product of neomycin phosphotransferase activity. The average NPTIIenzyme level determined for CaMV(35S) (pMON273) plants was 3.6 timeshigher than that determined for CaMV(19S) (pMON203 & 204 ) plants.

    ______________________________________                                        QUANTITATION OF NPTII ACTIVITY LEVELS                                         IN pMON203, pMON204, AND pMON273 PLANTS                                                 Plant    Relative                                                   Construct Number   NPTII Activity.sup.a                                                                         Average                                     ______________________________________                                        pMON203   4283     499,064        398,134                                     pMON203   4248     297,204        356,203                                     pMON204   4275     367,580        314,273                                     pMON204   4280     260,966                                                    pMON273   3350     1,000,674      1,302,731                                   pMON273   3271     1,604,788                                                   ##STR5##                                                                     ______________________________________                                         .sup.a Numbers represent quantitation of NPT assay. Values were obtained      by scintillation counting of .sup.32 PNPTII spots on the PE81 paper used      in the NPT assay as previously described.                                

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I claim:
 1. A differentiated dicotyledonous plant comprising plant cellscontaining a chimeric gene which comprises a promoter from cauliflowermosaic virus, said promoter selected from the group consisting of aCaMV(35S) promoter free of CaMV protein-encoding DNA sequences and aCaMV(19S) promoter free of protein-encoding DNA sequences, and astructural sequence which is heterologous with respect to the promoter.2. A differentiated plant of claim 1 in which the promoter is theCaMV(35S) promoter.
 3. A differentiated plant of claim 1 in which thepromoter is the CaMV(19S) promoter.
 4. A differentiated dicotyledonousplant comprising plant cells containing in the plant genome a chimericgene which comprises a promoter from cauliflower mosaic virus, saidpromoter selected from the group consisting of a CaMV(35S) promoter anda CaMV(19S) promoter, and a DNA sequence which is heterologous withrespect to the promoter.
 5. A differentiated dicotyledonous plantregenerated from plant cells, said plant cells containing a chimericgene which comprises a promoter from cauliflower mosaic virus, saidpromoter selected from the group consisting of a CaMV(35S) promoter anda CaMV(19S) promoter, and a DNA sequence which is heterologous withrespect to the promoter.